Synthesis of 4-Methoxy-1, 3-Benzenediolylhydrazones and Evaluation of Their Anti-Platelet Aggregation Activity

In our present investigation, a series of novel 4-methoxy-1,3-benzenediolyl-hydrazones were designed and synthesized, and their ability to inhibit platelet aggregation was evaluated by adenosine diphosphate (ADP) and arachidonic acid (AA). The structures of the synthesized compounds were conﬁrmed by spectral data. Results demonstrated that the activities of all compounds excelled the positive drug Picotamide (25.1% inhibition rate) and seven compounds (PNN01, PNN03, PNN05, PNN07, PNN09, PNN12, and PNN14) have efficiently inhibited platelet aggregation even higher than Clopidogrel (37.6% inhibition rate) induced by AA. Among them, PNN07 (39.8% inhibition rate) was considered as the most potent analogue. Evaluation of cytotoxic activity of the compounds against L929 cell line revealed that none of the compounds have signiﬁcant cytotoxicity. Thus, diolylhydrazones derives are potential to be antiplatelet aggregation inhibitors and maybe working in AA-induced selectively.


Introduction
Cardiovascular diseases (CVDs) account for over 17 million deaths globally each year (30% of all deaths) and the number is expected to grow to 23.6 million by 2030 (1). Most CVD patients are suffering from atherosclerosis, one due to many external factors, including physical insufficient physical activities, inadequate eating, and tobacco intake, and some internal risk factors, including diabetes, hypertension, and metabolic syndrome and so on (2). The rupture of atherosclerotic plaques and intracavitary thrombosis is the most common mechanism of most acute coronary syndromes and sudden coronary death, despite continued advances have been made in pharmacy and surgical treatments (3).
Platelet is a key participants in atherothrombosis (4, 5) due to their capacity to adhere to the injured blood vessel wall and amplify the procoagulant response (6, 7). In this process, a proaggregant signaling cascade is eventually triggered by plaque rupture, resulting in full platelet activation that leads to further aggregation and exposure of a procoagulant surface that enables the formation of a fibrin-rich hemostatic plug (8). In general, antithrombotic drugs, antiplatelet agents (eg. clopidogrel, aspirin®, tirofiban), are also the primary treatment options for these diseases.
However, major limitation including interindividual variability, increased risk of bleeding, neutropenia, thrombocytopenia, and drug resistance (9-13) need better and therapeutic drugs and in the same prompt the development of novel antiplatelet agents.
Picotamide (Figure 2 left), a novel antiplatelet agents, is a dual inhibitor of both TxA 2 receptors and TxA 2 synthase. In addition, in-vitro studies using rings of rat aorta demonstrated that Picotamide was unable to suppress PGI 2 , which is released from the endothelium and is generally inhibited by COX-1. This kind of dual action makes it possible to enhance therapeutic efficacy in the prevention of thrombosis, including inhibition of platelet aggregation and accumulation of antiaggregatory prostaglandins (PGI 2 and PGD 2 ) (14). Unfortunately, although it is safer than aspirin but still has a short halflife and weaker activity. However, it is these outstanding features of Picotamide that sparked our great interest so that over the past two decades, our research group has made many attempts to modify the structure of Picotamide based on the concept of bioisosterism. We envisioned replacing the two amide components in Picomaide with two ester components or two sulfonamide components and replacing the two 3-pycolyl groups in picotamide with two substituted phenyl groups in the same time. And we previously demonstrated a successful application of such analogues of Picomaide and showed that the derivatives exhibit excellent inhibitory effects in-vitro induced by ADP and AA (Figure 2 A, B, C) (15).
Several articles have been reported that N-acylheteroaryl hydrazones (NAH) had already become platelet aggregation inhibitors and other bioactive agents. Cunha et al., synthesized N-phenylpyrazolyl moiety which has the NAH chain exhibiting inhibitory effect on AA-, collagen, and ADP-induced platelet aggregation. So it represented a novel family of anti-platelet agents. Silva et al. in 2004 suggested that the hydrazone and acylhydrazone moieties demonstrate a subunit which stabilize free radicals that mimicking bis-allyl fragment of unsaturated fatty acids such as arachidonic acid (AA). Furthermore, these fragments have an important role as pharmacophore cores with anti-infammatory, anti-nociceptive, and antiplatelet aggregation activity (Figure 2 D) (16, 17).
Bezerra-Neto et al., ( Figure 2E) (18) have previously described the N-acylhydrazone subunit as a pharmacophore group for analgesic, anti-inflammatory, and anti-platelet properties, and it is also considered as a privileged structure Figure 1. Our previous works: A. One of the most potent compounds of 4-methoxy-1,3-benzenedicarboxamides series. B. The most potent compound of 4-methoxy diphenyl isophthalates series. C. One of the most potent compounds of 4-methoxy-1,3benzenedisulfonamides series. thrombocytopenia, and drug resistance (9-13) need better and therapeutic drugs and in the same prompt the development of novel antiplatelet agents.
Picotamide (Figure 2 left), a novel antiplatelet agents, is a dual inhibitor of both phenyl groups in the same time. And we previously demonstrated a successful application of such analogues of Picomaide and showed that the derivatives exhibit excellent inhibitory effects in-vitro induced by ADP and AA (Figure 2 A, B, C) (15).
Several articles have been reported that N-acylheteroaryl hydrazones (NAH) had already become platelet aggregation inhibitors and other bioactive agents. Cunha et al., synthesized N-phenylpyrazolyl moiety which has the NAH chain exhibiting inhibitory effect on AA-, collagen, and ADP-induced platelet aggregation. So it represented a novel family of anti-platelet agents. Silva et al. in 2004 suggested that the hydrazone and acylhydrazone moieties demonstrate a subunit which stabilize free radicals that mimicking bis-allyl fragment of unsaturated fatty acids such as arachidonic acid (AA). Furthermore, these fragments have an important role as pharmacophore cores with anti-infammatory, anti-nociceptive, and anti-platelet aggregation activity (Figure 2 D) (16, 17).
Bezerra-Neto et al., ( Figure 2E) (18) have previously described the N-acylhydrazone subunit as a pharmacophore group for analgesic, anti-inflammatory, and anti-platelet properties, and it is also considered as a privileged structure in the design of new bioactive compounds (19,20). Coquelet et al. have reported some acylhydrazone derivatives as inhibitors of platelet cyclooxygenase (COX) and lipooxigenase (LOX) blocking the transformation of arachidonic acid to its proaggregatory metabolites. In light of the finding of this study, Barreiro et al., in a series of studies, managed to develop some new hydrazones which effectively inhibited platelet aggregation with selective inhibitory activity toward platelet aggregation induced by arachidonic acid (AA). They found active group of hydrazones with arylsulfonate acylhydrazone, phenothiazine-1-acylhydraz-one, N-substituted-phenyl-1,2,3-triazole-4-acylhyd razone, and pyrazolylhydrazone structures. Although a diverse group of derivatives have been found to exhibit anti-platelet activity, in these studies they share a preserved structural backbone that is two (hetero) aromatic ring systems linked by a hydrazone bond ( Figure  2F) (21).
Moreover, in a successful study, indole ring is demonstrated as a structural moiety which has anti-platelet aggregation effect. Indole-3carbinol, a natural compound found in cruciferous vegetables, is known to have anti-platelet and anti-thrombotic activities in-vitro and in-vivo. Indole-3-carbinol has been shown to inhibit collagen-induced platelet aggregation in human platelet-rich plasma (PRP) in dose-dependent manner ( Figure 2G  Encouraged by these results of our previous studies on the biological properties of Picotamide's derivatives and based on the above mentioned reports, in this investigation, nineteen derivatives of 4-methoxy-1,3-benzenediolyl hydrazones bearing two hydrazone moiety compounds as potential antiplatelet agents were rationally designed and synthesized (Scheme 1.).
. blocking the transformation of arachidonic acid to its proaggregatory metabolites. In light of the finding of this study, Barreiro et al., in a series of studies, managed to develop some new hydrazones which effectively inhibited platelet aggregation with selective inhibitory activity toward platelet aggregation induced by arachidonic acid (AA). They found active group of hydrazones with arylsulfonate acylhydrazone, phenothiazine-1-acylhydraz-one, N-substituted-phenyl-1,2,3-triazole-4acylhydrazone, and pyrazolylhydrazone structures. Although a diverse group of derivatives have been found to exhibit antiplatelet activity, in these studies they share a preserved structural backbone that is two (hetero) aromatic ring systems linked by a hydrazone bond ( Figure 2F) (21).
Moreover, in a successful study, indole ring is demonstrated as a structural moiety which has anti-platelet aggregation effect. Indole-3carbinol, a natural compound found in cruciferous vegetables, is known to have antiplatelet and anti-thrombotic activities in-vitro and in-vivo. Indole-3-carbinol has been shown to inhibit collagen-induced platelet aggregation in human platelet-rich plasma (PRP) in dosedependent manner ( Figure 2G) (22).
Encouraged by these results of our previous studies on the biological properties of Picotamide′s derivatives and based on the above mentioned reports, in this investigation, nineteen derivatives of 4-methoxy-1,3-benzenediolyl hydrazones bearing two hydrazone moiety compounds as potential antiplatelet agents were rationally designed and synthesized (Scheme 1.).

Materials and instruments
All chemical reagents were purchased from Aladdin Industrial Corporation (P.R. China), Energy Chemical (P.R. China), and Tianjin Hengshan (P.R. China) and used without further purification. The reagents of cell viability were purchased from Beyotime Biotechnology (P.R. China). Other biological reagents arachidonic acid (AA) was purchased from Sigma and also, adenosine diphosphate (ADP) was purchased from Solarbio life sciences. The melting points were determined with a Kofler micro melting point apparatus and were uncorrected. Nuclear magnetic resonance ( 1 H NMR and 13 C NMR) spectra were recorded on a Bruker 400 MHz spectrometers (Bruker, Rheinstetten, Germany), pick positions are illustrated in parts per million (d) in DMSO-d 6 solution and TMS (0.05% v/v) as internal standard, and coupling constant values (J) are given in Hertz. Signal multiplicities are reported by: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), and brs (broad signal). For NMR spectral data assignments, the atom numbering of compounds is depicted in Scheme 2. Analytical thin-layer chromatography (TLC) was performed with Merck silica gel plates and visualized with UV irradiation (254 nm). High-resolution mass spectra (HRMS) Scheme 1. The concept of target compounds.

Materials and instruments
All chemical reagents were purchased from Aladdin Industrial Corporation (P.R. China), Energy Chemical (P.R. China), and Tianjin Hengshan (P.R. China) and used without further purification. The reagents of cell viability were purchased from Beyotime Biotechnology (P.R. China). Other biological reagents arachidonic acid (AA) was purchased from Sigma and also, adenosine diphosphate (ADP) was purchased from Solarbio life sciences. The melting points were determined with a Kofler micro melting point apparatus and were uncorrected. Nuclear magnetic resonance ( 1 H NMR and 13 C NMR) spectra were recorded on a Bruker 400 MHz spectrometers (Bruker, Rheinstetten, Germany), pick positions are illustrated in parts per million (d) in DMSO-d6 solution and TMS (0.05% v/v) as internal standard, and coupling constant values (J) are given in Hertz. Signal multiplicities are reported by: s (singlet), d (doublet), t (triplet), q (quadruplet), m (multiplet), and brs (broad signal). For NMR spectral data assignments, the atom numbering of compounds is depicted in Scheme 2. Analytical thin-layer chromatography (TLC) was performed with Merck silica gel plates and visualized with UV irradiation (254 nm). High-resolution mass spectra (HRMS) were recorded on an Agilent 6520B UPLC-Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Melting points were obtained by an Electrothermal 9100 apparatus and are uncorrected. The IR spectra were taken by a PerkinElmer 843 spectrometer with KBr as diluent.

General procedure for the preparation of 4-methoxyisophthalic acid (5):
Intermediates 2-5 were prepared according to the literature method (28)
were recorded on an Agilent 6520B UPLC-Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Melting points were obtained by an Electrothermal 9100 apparatus and are uncorrected. The IR spectra were taken by a PerkinElmer 843 spectrometer with KBr as diluent.

In-vitro evalution of anti-platelet aggregation activity
The in-vitro antiplatelet aggregation activities of nineteen derivatives of 4-methoxy-1,3-benzenedicarboxamides were measured by the turbidimetric method of Born and Cross (23). Blood was collected from anesthetized rats by venous puncture using syringes into tubes containing 3.8% sodium citrate (1:9, v/v). Platelet aggregation was assessed in platelet-rich plasma (PRP), obtained by centrifugation of citrated whole blood at room temperature for 10 min (500-800 rpm). The aggregation rate was measured by platelet aggregation analyzer after stimulation with AA (5 μM) and ADP using platelet-poor plasma (PPP) set to zero. The PPP was obtained by centrifugation of PRP at room temperature for 15 min (3000 rpm). A solution of the compounds (1.3 μmol/L) in DMSO (5 μL) was added into PRP (200 μL), and the same volume of DMSO have no significant effect on the platelet aggregation). After incubating for 2 min, the platelet aggregation was assessed and the percentage inhibition of platelet aggregation was calculated and the aggregation was monitored for 5 min. DMSO (0.5% v/v) was used as negative control and Picotamide and Clopidogrel as positive drugs. The results were expressed as mean±SD of 3 independent experiments. The platelet aggregation inhibition (%) was calculated by the following formula: Inhibition % = (1-D/S) × 100%; where D = platelet aggregation in the presence of test compounds, and S = platelet aggregation in the presence of solvent. The primary screening data for all compounds (1.3 μM) in-vitro activities on antiplatelet aggregation of the synthesized compounds are given in Table 1. The Statistical analysis was sodium hydroxide using tetrabutylammonium bromide (TBAB) as catalyst to produce 2-methoxy-5-methylacetophen-one 4 in 78.5% yield. Oxidation of the methyl and acetyl groups in compound 4 afforded 4-methoxyisophthalic acid 5 which can be intermediates 7 and products were all dissoluble in ethanol even in refluxed so their separation was difficult. Thus, glacial acetic acid was used as solvent and heated to complete the reaction.    162.38, 159.83, 146.81, 135.02, 134.60, 133.72, 132.53, 129.93, 129.28, 128.55, 125.62, 123.90, 118.25, 117.42, 56.77

N ′ 1 -( ( E ) -2 -h y d r o x y b e n z y l i d e n e ) -N ′ 3 -( 2 -h y d r o x y b e n z y l i d e n e ) -4methoxyisophthalohydrazide (PNN03)
Yield 65

Chemistry
The synthetic procedures to prepare these desired 4-methoxy-1,3-benzenediolyl hydrazones are shown in Scheme 2 4-Methylphenol 1 was used as starting material. Acetylation of 1 with acetic anhydride gave 4-methylphenyl acetate 2 which was subjected to Fries rearrangement to 5-methyl-2-hydroxyacetophenone 3 in the presence of AlCl 3 . The crude product yield was near 90% over two steps. Compound 3 was reacted with dimethyl sulfate in aqueous sodium hydroxide using tetrabutylammonium bromide (TBAB) as catalyst to produce 2-methoxy-5-methylacetophen-one 4 in 78.5% yield. Oxidation of the methyl and acetyl groups in compound 4 afforded 4-methoxyisophthalic acid 5 which can be isolated in the pure state by recrystallization from ethanol. Dimethyl-4methoxyisophthalate 6 was obtained by reacting 5 in the presence of methanol with thionyl chloride as catalyst. The key intermediates 4-methoxyisophthalohydrazide 7 were obtained by hydrazinolysis of 6 and in 85.2% yield, using hydrazine monohydrate 80% in ethanol. The final 4-Methoxy-1,3-benzenediolylhydrazone derivatives 9 were collected by condensing the hydrazide intermediates with the proper aromatic aldehydes in glacial acetic acid as the solvent refluxed for several minutes, in good yields. Synthesis of Schiff bases was performed in glacial acetic acid. This reaction in many cases was straightforward, but the intermediates 7 and products were all dissoluble in ethanol even in refluxed so their separation was difficult. Thus, glacial acetic acid was used as solvent and heated to complete the reaction.

Biological Evaluation In-vitro evalution of anti-platelet aggregation activity
The in-vitro antiplatelet aggregation activities of nineteen derivatives of 4-methoxy-1,3benzenedicarboxamides were measured by the turbidimetric method of Born and Cross (23). Blood was collected from anesthetized rats by venous puncture using syringes into tubes containing 3.8% sodium citrate (1:9, v/v). Platelet aggregation was assessed in plateletrich plasma (PRP), obtained by centrifugation of citrated whole blood at room temperature for 10 min (500-800 rpm). The aggregation rate was measured by platelet aggregation analyzer after stimulation with AA (5 μM) and ADP using platelet-poor plasma (PPP) set to zero. The PPP was obtained by centrifugation of PRP at room temperature for 15 min (3000 rpm). A solution of the compounds (1.3 μmol/L) in DMSO (5 μL) was added into PRP (200 μL), and the same volume of DMSO without test compound was added to a reference sample (according to a preexperiment, 5 μL of DMSO appears to have no significant effect on the platelet aggregation). After incubating for 2 min, the platelet aggregation was assessed and the percentage inhibition of platelet aggregation was calculated and the aggregation was monitored for 5 min. DMSO (0.5% v/v) was used as negative control and Picotamide and Clopidogrel as positive drugs.
The results were expressed as mean±SD of 3 independent experiments. The platelet aggregation inhibition (%) was calculated by the following formula: where D = platelet aggregation in the presence of test compounds, and S = platelet aggregation in the presence of solvent. The primary screening data for all compounds (1.3 μM) in-vitro activities on antiplatelet aggregation of the synthesized compounds are given in Table  1. The Statistical analysis was performed with ANOVA followed by Tukey′s test.

Cytotoxicity effect on L-929 cells (Figure 3)
Mouse fibroblast cells (L929) were chosen to evaluate the in-vitro cytotoxicity of the materials and the drugs via Cell Counting Kit-8 (CCK-8) assays. L929 was cultivated in a humidified 5% carbon dioxide atmosphere at 37 °C on 96-well microplates, with 1×10 4 cells per well immersed in complete growth medium. The cells with 100 mL of RPMI-1640 per well were allowed to be attached for 24 h. Subsequently, the cells were then exposed to target compounds at a range of concentrations at 37 °C for 48 h. Target compounds concentration of 10 and 100 μmol L -1 were added to L929 cells. After incubation for 48 h, the medium was removed and replaced with 100 μL of fresh complete medium of RPMI-1640. Then, CCK-8 solution was added to the Data are expressed as the means±standard errors of the means. Statistical differences between the experimental and control groups were evaluated by analysis of variance followed by the Tukey test. * p < 0.05 vs. the control group; † p < 0.01 vs. Picotamide. ** p < 0.05 vs. Picotamide; ADP, adenosine diphosphate; AA, arachidonic acid. 96-well plates at 10 μL per well and incubated for a further 30 min, and the absorbance at 450 nm was measured on a microplate reader (Bio-Tek FLx800 fluorescence microplate reader). This process was repeated eight times in parallel. The results are expressed as the relative cell viability (%) with respect to control wells, calculated as follows (24).

Chemsitry
The synthetic pathway is disclosed in Scheme 2. Final desired N-acylhydrazones were prepared through a classic imine formation reaction between 4-methoxy-isophthalohydrazide and different aromatic aldehydes. The 1 H NMR and HR ESI-MS data of compounds approved the exact structures. In the 1 H NMR spectra of these compounds, the existence of two singlets at 11.0 -12.0 ppm was assigned to hydrazide C = ONH. Singlet signal at 8.2 -8.9 ppm was assigned to N=CH. Molecular mass of all the derivatives was detected by high-resolution electron spray ionization mass spectrometry (HR ESI-MS) as M+1 relating to hydrogen of the intact molecules.
The acylhydrazone structures gave two sets of signals in NMR spectra whose intensities depend on solvent and the pair of signals coalesces on warming the NMR tube. The existence of the carbonyl oxygen atom and the imine nitrogen atom in N-acylhydrazones indicates geometrical isomers (E/Z). Syakaev et al. also noticed that derivatives of acylhydrazones from aromatic aldehydes showed two rotamers (cis and trans) due to the N-C (O) bond (25,26).
In our study, the 1 H NMR spectra at room temperature for some compounds of target compounds represented E/Z isomerization which was related to CH = N double bonds. Right after dissolution of the synthesized compounds in DMSO-d 6 , the double signals of E C=N /Z C=N conformers are registered by NMR. The two conformers show two sets of signals at different chemical shifts, and integration of the two sets of signals indicates that the E C=N conformer is the predominant conformer. Based on the earlier report that N-acylhydrazones derived from aromatic aldehydes in solution remained mostly in the E form because of the hindered rotation on the imine bond (27), we thought that E geometry in our cases. then exposed to target compounds at a range of concentrations at 37 °C for 48 h. Target compounds concentration of 10 and 100 μmol L -1 were added to L929 cells. After incubation for 48 h, the medium was removed and replaced with 100 μL of fresh complete medium of RPMI-1640. Then, CCK-8 solution was added to the 96-well plates at 10 was repeated eight times in parallel. The results are expressed as the relative cell viability (%) with respect to control wells, calculated as follows (24).

Chemsitry
The synthetic pathway is disclosed in Scheme 2. Final desired N-acylhydrazones were prepared through a classic imine formation reaction between 4-methoxy-isophthalohydrazide and different aromatic aldehydes. The 1 H NMR and HR ESI-MS data of compounds approved the exact structures. In the 1 H NMR spectra of these compounds,

Anti-platelet aggregation activity
The in-vitro antiplatelet activities of all synthesized compounds were assayed on rat platelet rich plasma (PRP) by using the Born′s turbidimetric method. ADP and Arachidonic acid (AA) were employed as inducers of platelet aggregation; both Picotamide and Clopidogrel were used as the positive controls. Inhibition rate values were calculated for the ability of the test compounds. The antiplatelet aggregation activity of the derivatives is listed in Table 1. Data show that the majority of the derivatives inhibited AA-induced platelet aggregation more effectively than the aggregation induced by ADP and also, some compounds showed inhibitory effects compared to clopidogrel. Among the tested compounds, derivatives PNN01, PNN03, PNN05, PNN07, PNN09, PNN12 and PNN14 showed high inhibition values and PNN07 was the most potent compound with inhibition value of 39.8% induced by AA. Among all the target compounds, on their substituted benzene rings, based on the activity data, it could be suggested that the most active compounds were among the electron-donating group structures. Hence, electronic properties of the aromatic ring may possibly contribute to the activity of the compounds and with one substitute is more potent than two. Just like the effects of electronic factors, there is no obvious rule for the size of substitutes on the anti-platelet aggregation activity.

Cytotoxicity effect on L-929 cells
The calculated cytotoxicity effect on L-929 cells of seven compounds PNN01, PNN03, PNN05, PNN07, PNN09, PNN12 and PNN14 are given in Figure 3. The data analysis demonstrated that at lower concentration of 10 μmol/L, three compounds PNN01, PNN03, and PNN07 have lower effect on L-929 cells, and the effect of PNN03 is close to the control drug Picotamide. At the concentration of 100 μmol/L, the effects of three compounds are better than those of Picotamide.

Conclusion
We have rationally designed and synthesized nineteen analogues of Picotamide PNN01-PNN19. It was found that all the diolylhydrazones derivatives are having platelet aggregation inhibitory effect. In addition, comparing the activity of compounds against platelet aggregation induced by AA shows that compounds PNN01, PNN03, PNN05, PNN07, PNN09, PNN12, and PNN14 have significant antiplatelet aggregation and even higher than Clopidogrel (37.6%). However, when induced by ADP all the compounds showed little activities and lower than Picotamide (40.4%). So, the tested derivatives selectively inhibited platelet aggregation induced by AA with very good inhibition rates values. Among them, compound PNN07 with inhibition value of 39.8% proved to be the most potent derivative of this series. Evaluation of cytotoxic activity of the compounds against L929 cell line revealed that none of the compounds have significant cytotoxicity. Thus, we confirmed that diacylhydrazone obtains the ability of antiplatelet aggregation and maybe works in AA-induced selectively. Further study is worthy to be done for the many reasons.